Graves'disease (GD) is a syndrome caused by as yet unidentified genetic and environmental factors where the thyroid becomes overactive because antibodies (TSI) directed against the thyrotropin receptor (TSHR) drive excess hormone production. GD is also associated with the inflammation, accumulation of hyaluronan (HA), and remodeling of orbital connective tissue. This process is known as thyroid-associated ophthalmopathy (TAO) for which no effective treatment currently exists. A major impediment to our understanding of TAO is the absence of a pre-clinical animal model. We have discovered previously that orbital fibroblasts from patients with TAO exhibit a unique inflammatory phenotype and over-express the insulin-like growth factor-1 receptor (IGF-1R). IGF-1R-activating antibodies (GD-IgGs) are found in virtually all patients with GD. When they bind to this receptor, T cell chemoattractants and HA are synthesized by TAO fibroblasts. We hypothesize that these events underlie lymphocyte infiltration and expansion of the orbital tissue. We have now discovered that GD is also associated with an increased frequency of IGF-1R+ T and B cells and this phenotype conveys a proliferative and survival advantage. Monozygotic (MZ) twins discordant for GD are also discordant for the IGF- 1R+ lymphocyte skew, suggesting an environmental cause. We have also now discovered that patients with GD have increased numbers of circulating CD34+ fibroblast precursors called fibrocytes. Moreover we find fibrocytes infiltrating the TAO orbit and expressing high levels of TSHR and IGF-1R. In orbital fibroblasts and thyroid epithelium, TSHR and IGF-1R form a physical complex and IGF-1R mediates aspects of TSHR signaling. We have succeeded in cloning an adenovirus encoding an IGF-1R1-GFP fusion protein. When female BALB/c mice are immunized with it, they manifest orbital inflammation and produce GD-IgG. We have also implanted TAO fat into SCID NOD3-/- mice and shown that TAO fibrocytes home to that tissue. We hypothesize that IGF-1R represents the second critical auto-antigen in GD and is necessary with TSHR to generate authentic disease. We now propose studies to test our central hypothesis. Specific aim 1) To characterize profiles of CD4+ IGF-1R+ T cells, CD19+IGF-1R+ B cells, and fibrocytes from patients with GD, determine whether they change longitudinally or predict development of TAO, determine whether IGF-1R+ B cells selectively produce pathogenic Abs such as TSI and GD-IgG, and examine additional MZ twins discordant for clinical GD. Specific aim 2) To characterize the interaction between IGF-1R and TSHR by cloning IGF-1R2 mutants and performing yeast two-hybrid analysis. Specific aim 3) To continue exploiting two mouse models of GD created by 1) implanting orbital fat into SCID NOD3-/- mice and 2) double-immunizing mice with adenoviruses encoding IGF-1R and TSHR. We believe that important insights into disease pathogenesis and identification of novel therapeutic targets will emerge from these studies. PUBLIC HEALTH RELEVANCE: Thyroid-associated ophthalmopathy (TAO) is the sight-threatening orbital component of Graves'disease, a syndrome where the thyroid gland becomes over-active. There are currently no effective therapies for TAO because we lack an experimental animal model to study disease mechanisms and test therapies. In this proposal, we identify abnormalities in human immune system that underlie development of TAO, and apply these insights to animal models in which treatments can be developed.